Receptor tyrosine kinases of the ErbB family play important roles in the development and progression of various types of human carcinoma. Elucidation of the physiological regulation of a prototypical member of the family, epidermal growth factor (EGF) receptor (EGFR/ErbB1), and other ErbBs, is the key to understanding the mechanisms causing their oncogenic activation. Growth factor binding to the EGFR results in initiation of a myriad of signal transduction processes. Activation of the mitogen-activated protein kinase 1/2 (MAPK/ERK1/2) is the major signaling pathway triggered by EGFR, which determines the ultimate outcome of the EGFR signaling. Recent studies revealed that EGFR signaling is not limited by signaling at the cell surface, but, rather, all components of the pathway move within the cell and may associate with and function in various intracellular compartments. The activated EGFR is rapidly internalized into endosomes and sorted to lysosomes for degradation. There are numerous bi-directional cross-talks between signaling and endocytic machineries. It has been proposed that endocytosis is necessary for MAPK activation, although this issue remains controversial. The overall goal of this proposal is to reconstitute the signaling process in space and time in cancer cells and to integrate the new experimental data into a computational model of the dynamics of the EGFR-MAPK pathway. Firstly, the components of the MAPK signaling pathway will be tagged with various fluorescent proteins (XFPs) and stably expressed at physiological levels in human carcinoma cells, in which the corresponding endogenous protein had been knocked-down by vector-mediated RNA interference. The patterns of the dynamic localization and trafficking of XFP-tagged proteins involved in ERK1/2 activation will then be analyzed in these new cell lines using quantitative fluorescence microscopy. Secondly, using a combination of RNA interference, live-cell fluorescence imaging, multi-chromophore fluorescence resonance energy transfer microscopy and biochemical techniques, the mechanisms of the formation of the multi-protein complexes during MAPK activation and the localization of these complexes will be investigated. Thirdly, a systematic analysis of the role of endocytosis and endosomes in the regulation of EGFR signaling to MAPK will be performed in a range of human cancer cell lines expressing various levels of EGFR. Using the experimental data describing the spatial and temporal overlap of the components of the EGFR-MAPK pathway, their diffusion rate parameters, routes of trafficking and protein-protein interactions, a new computational model of spatial and temporal organization of EGFR-MAPK signaling will be generated, experimentally validated and used to predict the effects of different EGFR expression levels, concentrations of EGFR ligands and various perturbations of the system on the intensity and duration of MAPK signaling. PUBLIC HEALTH RELEVANCE: The EGF receptor has become the major prognostic and diagnostic marker and an important therapeutic target in human carcinoma. However, the benefits from using the EGF receptor as a marker and a therapeutic target are currently highly limited. The studies proposed here will enhance our understanding of the regulation of EGF receptors in cancer cells and should help in developing new strategies of down regulating the activity of EGF receptors and similar receptors in cancer cells and may also reveal new potential prognostic markers and therapeutic targets.